Thermodynamic Criterion for the Conformation of P1 Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases
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- 作者：M. A. Qasim ; Stephen M. Lu ; Jinhui Ding ; Katherine S. Bateman ; Michael N. G. James ; Stephen Anderson ; Jikui Song ; John L. Markley ; Philip J. Ganz ; Charles W. Saunders ; and Michael Laskowski ; Jr.
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：June 1, 1999
- 年：1999
- 卷：38
- 期：22
- 页码：7142 - 7150
- 全文大小：129K
- 年卷期：v.38,no.22(June 1, 1999)
- ISSN：1520-4995
文摘
Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) areall standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to adifferent inhibitor family. Therefore, all three have the same canonical conformation in their combiningloops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than itsAla45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly,turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant(the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differencesare within the ±400 cal/mol bandwidth (expected from the experimental error), one can conclude that thesystem is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Alapair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificitypocket will be identical. This is confirmed, within the experimental error, by the available X-ray structuresof complexes of bovine chymotrypsin A
with eglin c (lacb) and with turkey ovomucoid third domain(1cho). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsininhibitor (Kunitz) (1mtn and 1cbw) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain(1hja), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikinglydifferent. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more stronglythan its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). Incontrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less stronglythan its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In thiscase, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether theconformation of a P1 side chain in the complex matches that of an already determined one is at hand.Such a criterion may be useful in reducing the number of required X-ray crystallographic structuredeterminations. More importantly, the criterion can be applied to situations where direct determination ofthe structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ sidechain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements,the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domainvariants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expectthat in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeplyinserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complexwith chymotrypsin.
with eglin c (lacb) and with turkey ovomucoid third domain(1cho). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsininhibitor (Kunitz) (1mtn and 1cbw) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain(1hja), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikinglydifferent. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more stronglythan its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). Incontrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less stronglythan its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In thiscase, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether theconformation of a P1 side chain in the complex matches that of an already determined one is at hand.Such a criterion may be useful in reducing the number of required X-ray crystallographic structuredeterminations. More importantly, the criterion can be applied to situations where direct determination ofthe structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ sidechain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements,the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domainvariants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expectthat in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeplyinserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complexwith chymotrypsin.